title: The basic cleft of RPA70N binds multiple checkpoint proteins, including RAD9, to regulate ATR signaling.
abstract: ATR kinase activation requires the recruitment of the ATR-ATRIP and RAD9-HUS1-RAD1 (9-1-1) checkpoint complexes to sites of DNA damage or replication stress. Replication protein A (RPA) bound to single-stranded DNA is at least part of the molecular recognition element that recruits these checkpoint complexes. We have found that the basic cleft of the RPA70 N-terminal oligonucleotide-oligosaccharide fold (OB-fold) domain is a key determinant of checkpoint activation. This protein-protein interaction surface is able to bind several checkpoint proteins, including ATRIP, RAD9, and MRE11. RAD9 binding to RPA is mediated by an acidic peptide within the C-terminal RAD9 tail that has sequence similarity to the primary RPA-binding surface in the checkpoint recruitment domain (CRD) of ATRIP. Mutation of the RAD9 CRD impairs its localization to sites of DNA damage or replication stress without perturbing its ability to form the 9-1-1 complex or bind the ATR activator TopBP1. Disruption of the RAD9-RPA interaction also impairs ATR signaling to CHK1 and causes hypersensitivity to both DNA damage and replication stress. Thus, the basic cleft of the RPA70 N-terminal OB-fold domain binds multiple checkpoint proteins, including RAD9, to promote ATR signaling.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, 613 Light Hall, 23rd at Pierce Avenue, Nashville, TN 37232, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Xu
authors.forename: Sivaraja
authors.initials: S
authors.lastname: Vaithiyalingam
authors.forename: Gloria G
authors.initials: GG
authors.lastname: Glick
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Mordes
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Chazin
authors.forename: David
authors.initials: D
authors.lastname: Cortez
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004249:DNA Damage; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology*
chemical_list: C521194:ATRIP protein, human; D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; C497167:RPA1 protein, human; D051716:Replication Protein A; C069930:rad9 protein; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases